A high-performance liquid chromatography method with pre-column derivatization for the simultaneous determination of reduced glutathione, carnosine and taurine

Abstract

A new gradient reversed-phase HPLC method with pre-column derivatization was developed and validated for simultaneous determination of reduced glutathione, carnosine and taurine in new pharmaceutical eye drops. Chromatographic separation was achieved with a fused core analytical column Supelco Ascentis Express C18 (100 mm × 4.6 mm, 2.7 μm). A 30 mM triethylamine in acetonitrile with 30 mM triethylamine aqueous solution (pH 2.5, adjusted with glacial acetic acid) was used in a gradient elution mode at a flow rate of 0.8 mL min⁻¹. The column temperature was maintained at 30 °C, the injection volume of the derivatized sample was 5 μL and the detection wavelength was 375 nm. The tested validation parameters included system suitability, accuracy, precision, linearity, LOD, LOQ and sample solution stability. The standard calibration curves showed good linearity with correlation coefficients r > 0.997 for all compounds. The mean percentage recoveries obtained for reduced glutathione, carnosine and taurine were 99.61, 99.38 and 99.43%, respectively. Precision was <2% for retention times and peak areas. The applicability of this method was demonstrated by the analysis of a multicomponent eye drop formulation containing reduced glutathione, carnosine and taurine.